RESUMO
Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3' mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in â¼6 min, with â¼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from â¼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.
Assuntos
Leucócitos Mononucleares/metabolismo , Transcriptoma , Linhagem Celular , Feminino , Humanos , Leucócitos Mononucleares/química , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula ÚnicaRESUMO
Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.
Assuntos
Haplótipos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Análise de Sequência de DNA/métodos , DNA/genética , Genoma Humano , Variação Estrutural do Genoma , Células Germinativas , Humanos , Conformação de Ácido Nucleico , Proteínas de Fusão Oncogênica/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100,000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.
